Myoglobin is one of the major proteins in skeletal and cardiac muscle. This oxygen binding lieme protein is comprised of a single polypeptide chain with a reported molecular weight of 17.8 kDa. Myoglobin's tertiary structure has been extensively studied, and it is reported that 75% or the main chain is folded in an alpha helical conformation (Edmundson A. B., Biochem. Prop. 1968; 12, 41-52; Kagen L. J. (1973) In: F. Borek (Ed.), Myoglobin, Biochemical, Physiological, and Clinical Aspects. Columbia University Press, New York).
Although its precise physiological function remains controversial, it is known that its ability to combine reversibly to oxygen is reflective of its role in oxygen transport to the muscle cell. Normal human myoglobin levels in the blood range from 0 to 70 ng/ml.
Following muscle injury, both cardiac and skeletal serum levels of myoglobin are dramatically increased. Reflective of its small molecular size, myoglobin is able to translocate into the vascular system without processing in the lymphatic system. In certain health disorders and/or diseases serum myoglobin is known to be elevated. This elevation is thought to be caused by the release of myoglobin from damaged or necrotic muscle cells.
Acute Myocardial Infarction (AMI) affects millions of people each year, many of whom die because diagnosis or treatment was not available in time to save their lives. Studies have shown that if a correct diagnosis of AMI, and appropriate therapeutic intervention is performed within the first 6 hours after the onset of chest pain, the chances of survival are greatly increased. Thus, the goal of medical investigators has been to develop an analytical system which will identity those early indicators of this condition. In MI patients, blood, serum and plasma cardiac myoglobin levels are known to be above normal as early as 30 minutes after the onset of chest pain. In fact, in some MI patients, cardiac myoglobin levels may increased by 10 fold of that of a normal person over the course of the MI. Myoglobin is said to display a temporal release into the circulation. In MI patients, myoglobin levels may rise above normal within 2 hours, will reach peak serum levels in 6-9 hours, and will return back to normal levels by 24 to 36 hours after the onset of chest pain (Vaidya H. C., Laboratory Medicine, 1992; 23:306-310). Thus monitoring the rapid release of cardiac myoglobin can be used as an indicator of MI.
MI patients in the early stages of the disease (under 6 hours) are most often administered reperfusion treatment with streptokinase or TPA (tissue plasminogen activator) These thrombolytic agents act to restore blood flow in the occluded vessels. The serial measurement of myoglobin has proven useful in the monitoring of such treatments, since myoglobin levels peak approximately 45 minutes after successful reperfusion (Bllis A. K., et, al, Circulation, 1985; 72:639-647).
Several detection methods for myoglobin have been established, but each method has limitations which directly affect its clinical utility. The need for a non-invasive method of myoglobin detection was realized more than 25 years ago, and the earliest tests developed were radioimmunoassay (Stone M. J., et. al., J. Clin Invest., 1975; 56:1334-1339). In these assays the serum sample was combined with a radiolabelled myoglobin and an anti-myoglobin polyclonal antibody. The antibody was then precipitated with a second radiolabelled polyclonal antibody. The concentration of myoglobin in the sample was calculated based on the inverse of the amount of precipitated radioactivity. This method was limited as it required extremely skilled technicians, was time consuming and posed a radiation risk.
The latex agglutination method of myoglobin detection utilizes monoclonal antibodies directed to myoglobin, which have been immobilized on later particles (Chapelle J. P., et al. Clin. Chim. Acta. 1985; 145:143-150). These monoclonal antibodies combinc with serum myoglobin and form aggregates. The quantification of the aggregation is proportional to the myoglobin concentration. This assay is more rapid and practical than the RIA, but only semi-qualitative results are produced. This method was recently adapted into both the turbidometric (Turbitimer, Behring, Germany, Tuengler P., et al., Behring Inst. Mitt., 1988; 82:282-308) and the immunonephelometric system (NA Latex Myoglobin, Behring, Germany, Massoubre, C., Clin. Chim. Acta., 1991; 201:223-230). Both of these methods report low intra- and inter-assay variation. These methodologies are limited however, because of the analytical time required, inadequate specificity and the need for expensive analyzers.
Therefore, there remains a need for a monoclonal antibody that demonstrates high affinity and specificity for human myoglobin that can be used as a reagent in an immunoassay system to identity blood, serum or plasma levels of myoglobin in patients with cardiac muscle damage (e.g. myocardial infarction). Such an immunoassay system, can be used for diagnosing and quantifying myocardial necrosis and infarction according to the rapid format procedure disclosed in U.S. Pat. No. 5,290,678.